1. Field of the Invention
The present invention relates to optical scanning systems for examining biological material and, in particular, to filtering emission signals from fluorescently tagged biological materials that have been excited by lasers.
2. Related Art
Synthesized nucleic acid probe arrays, such as Affymetrix(copyright) GeneChip(copyright) synthesized probe arrays, have been used to generate unprecedented amounts of information about biological systems. For example, a commercially available GeneChip(copyright) array set from Affymetrix, Inc. of Santa Clara, Calif., is capable of monitoring the expression levels of approximately 6,500 murine genes and expressed sequence tags (EST""s). Experimenters can quickly design follow-on experiments with respect to genes, EST""s, or other biological materials of interest by, for example, producing in their own laboratories microscope slides containing dense arrays of probes using the Affymetrix(copyright) 417(trademark) or 427(trademark) Arrayers or other spotting devices. Analysis of data from experiments with synthesized and/or spotted probe arrays may lead to the development of new drugs and new diagnostic tools.
In some conventional applications, this analysis begins with the capture of fluorescent signals indicating hybridization of labeled target samples with probes on synthesized or spotted probe arrays. The devices used to capture these signals often are referred to as scanners. Due to the relatively small emission signals sometimes available from the hybridized target-probe pairs, the presence of background fluorescent signals, the high density of the arrays, variations in the responsiveness of various fluorescent labels, and other factors, care must be taken in designing scanners to properly acquire and process the fluorescent signals indicating hybridization. For example, U.S. Pat. No. 6,171,793 to Phillips, et al., hereby incorporated herein in its entirety for all purposes, describes a method for scanning probe arrays to provide data having a dynamic range that exceeds that of the scanner. As another example, U.S. patent application Ser. No. 09/681,819, filed Jun. 11, 2001 and hereby incorporated herein by reference in its entirety for all purposes, describes systems and methods for aligning grids on scanned images to provide appropriate pixel analysis. Nonetheless, there is a continuing need to improve scanner design to provide more accurate and reliable fluorescent signals and thus provide experimenters with more sensitive and accurate data.
High-density probe arrays allow higher throughput and other advantages as compared to probe arrays of lower densities. However, as the density of probes grows, the difficulties also generally increase of accurately and reliably distinguishing adjacent probes and collecting sufficient image information, i.e., pixels, specific to each probe. Similar difficulties emerge as the goal of higher throughput results in faster image scans. There thus is a need for methods and systems for scanning high-density probe arrays, possibly at higher scanning speeds, but yet preserving high sensitivity, accuracy, and reliability with respect to the acquisition of image pixels.
Systems, methods, and products to address these and other needs are described herein with respect to illustrative, non-limiting, implementations. Various alternatives, modifications and equivalents are possible. For example, certain systems, methods, and computer software products are described herein using exemplary implementations for analyzing data from arrays of biological materials produced by the Affymetrix(copyright) 417(trademark) or 427(trademark) Arrayer. Other illustrative implementations are referred to in relation to data from Affymetrix (copyright) GeneChip(copyright) probe arrays. However, these systems, methods, and products may be applied with respect to many other types of probe arrays and, more generally, with respect to numerous parallel biological assays produced in accordance with other conventional technologies and/or produced in accordance with techniques that may be developed in the future. For example, the systems, methods, and products described herein may be applied to parallel assays of nucleic acids, PCR products generated from cDNA clones, proteins, antibodies, or many other biological materials. These materials may be disposed on slides (as typically used for spotted arrays), on substrates employed for GeneChip(copyright) arrays, or on beads, optical fibers, or other substrates or media. Moreover, the probes need not be immobilized in or on a substrate, and, if immobilized, need not be disposed in regular patterns or arrays. For convenience, the term probe array will generally be used broadly hereafter to refer to all of these types of arrays and parallel biological assays.
In accordance with one preferred embodiment, an apparatus is described for scanning probe arrays. The apparatus includes an excitation filter that filters one or more excitation signals, and an emission filter that filters one or more emission signals. The excitation filter and the emission filter of this embodiment are linear-phase filters. The terms excitation signal and emission signal will be understood in this context to refer to sets of analog or digital values indicative of the power or other measure of all or portions of excitation beams and resulting emission beams, respectively. The terms excitation beam and emission beam are intended to have broad meanings, as described below. Thus, as one non-limiting example, a first emission signal may be generated by an emission detector (such as a photodiode) and represent fluorescent emissions from fluorophores excited by an excitation beam during a first scan of a portion of a probe array. As noted below, a scan may refer to relative movement of the excitation beam with respect to the probe array along a line or an arc. A second emission signal may represent emissions from the same or other fluorophores excited by the excitation beam during a second, or substantially simultaneous, scan of the same or other portion of the probe array.
In accordance with some preferred embodiments, an apparatus is described that includes an excitation beam provider, an emission signal detector, and a linear-phase emission filter. The excitation beam provider directs an excitation beam to a plurality of locations of a probe array, and the emission signal detector detects an emission signal from at least one location. The excitation beam provider may include an excitation source, such as a laser, as well as optical, mechanical, electrical, and other components (such as mirrors, scanning arms, or galvanometers, for example). The linear-phase emission filter filters the emission signal to provide a filtered emission signal having substantially symmetrical rise and fall edges. The term substantially symmetrical rise and fall edges refers to certain advantageous characteristics of the filtered waveforms, described in detail below with respect to FIGS. 7A-7E and 8A-E. These characteristics enable sampling of emission signals to be accomplished consistently irrespective of the scanning direction, and thus linear-phase emission signal filtering is particularly advantageous in bi-directional scanning applications. The word substantially is intended to connote that precise symmetry is not required and that the degree of symmetry generally depends on sampling method, sampling criteria, and other factors that will be appreciated by those of ordinary skill in the art in view of the detailed description below.
Apparatuses in accordance with these and other embodiments may also include an excitation signal detector that detects an excitation signal, and an excitation filter that provides a filtered excitation signal. The emission filter and the excitation filter may be matched with each other. Also, the emission filter may have a first delay function and the excitation filter a second delay function, and the apparatus may further include a delay compensator that compensate for any differences between the first and second delay functions.
In some implementations of these and other embodiments, the excitation filter may include a linear-phase filter that provides a filtered excitation signal having substantially symmetrical rise and fall edges. The emission filter and the excitation filter may both comprise a high-order Bessel filter, such as a sixth order, or an eleventh order, Bessel filter. The emission filter may normalize the emission signal to avoid aliasing errors, and may be, or include, a low-pass, anti-aliasing filter. One or more characteristics of the emission filter may be determined, at least in part, based on a scan rate or a resolution. A low-pass characteristic of the linear-phase emission filter may be determined, at least in part, based on applying the Nyquist criterion to a sampling rate.
The apparatus of these and other embodiments may also include a clock signal generator that digitizes the filtered emission signal, thereby providing a plurality of digitized emission values. Also included in the apparatus may be an excitation signal detector that detects an excitation signal indicative of the excitation beam, and an excitation filter that filters the excitation signal to provide a filtered excitation signal. In these implementations, the clock signal digitizes the filtered excitation signal, thereby providing a plurality of digitized excitation values. The emission filter and the excitation filter may be matched so that the plurality of digitized emission values are spatially correlated with the plurality of digitized excitation values. In some embodiments, the excitation beam is directed to the locations of the probe array so that dx/dt is substantially constant. The probe array may include a synthesized or a spotted probe array.
In accordance with other preferred embodiments, a method is described for analyzing molecules. The method includes (1) directing an excitation beam to a plurality of probe locations; (2) receiving an emission signal; (3) linear-phase filtering the emission signal to provide a filtered emission signal having substantially symmetrical rise and fall edges; and (4) analyzing molecules at one or more probe locations based, at least in part, on one or more values of the filtered emission signal. Also described in accordance with preferred embodiments is a method including detecting an emission signal from a probe array and filtering the emission signal to provide a filtered emission signal having substantially symmetrical rise and fall edges.
In yet other preferred embodiments, a noise compensation module is described having an excitation signal filter that filters at least a first and a second excitation signal and an emission signal filter that filter at least a first and a second emission signal. The excitation signal filter and the emission signal filter are linear-phase filters, the first excitation signal and corresponding first emission signal are derived from a scan in a first direction, and the second excitation signal and corresponding second emission signal are derived from a scan in a second direction that is opposite to the first direction. The excitation signal filter and the emission signal filter may be matched with each other, and both may include a high-order Bessel filter. Also described in accordance with other preferred embodiments is a method including filtering at least a first and a second excitation signal and filtering at least a first and a second emission signal. Filtering characteristics applied to the first and second excitation signals, and to the first and second emission signals, are linear-phase. The first excitation signal and corresponding first emission signal are derived from a scan in a first direction, and the second excitation signal and corresponding second emission signal are derived from a scan in a second direction that is opposite to the first direction.
Also described in accordance with various preferred embodiments is an apparatus having an emission signal detector and an emission filter. The detector detects an emission signal from at least one probe, and the emission filter filters the emission signal to provide a filtered emission signal having substantially symmetrical rise and fall edges. In further embodiments, a method is described having the steps of detecting an emission signal from at least one probe, and filtering the emission signal to provide a filtered emission signal having substantially symmetrical rise and fall edges.
The above embodiments and implementations are not necessarily inclusive or exclusive of each other and may be combined in any manner that is non-conflicting and otherwise possible, whether they be presented in association with a same, or a different, embodiment or implementation. The description of one embodiment or implementation is not intended to be limiting with respect to other embodiments and/or implementations. Also, any one or more function, step, operation, or technique described elsewhere in this specification may, in alternative implementations, be combined with any one or more function, step, operation, or technique described in the summary. Thus, the above embodiment and implementations are illustrative rather than limiting.